Ikhithi ye-RT-qPCR eqondile
Incazelo yekhithi:
◮ Ikhithi eqondile ye-RT-qPCR, isebenzisa i-Foreasy Reverse Transcriptase ne-Foreasy HS Taq DNA Polymerase eyakhiwe yi-Foregene, ene-react buffer eyingqayizivele, yenza le khithi ibe nokumelana okuqinile nokuhambisana, futhi ingahlola ngokuqondile ukusetshenziswa kwesistimu ye-Foregene Lysis njengesifanekiso.Le khithi ingasebenza ekutholeni ikhithi ye-COVID-19 nucleic acid kanye namanye amabhaktheriya e-pathogenic nucleic acid yokuthuthukiswa kwekhithi.
Incazelo
Ikhithi ye-RT-qPCR eqondile ihlinzeka ngebhafa ehlanganiswe ngokuhlukene ye-RT-qPCR kanye ne-enzyme esebenza kahle kakhulu.ingxube (i-Taq, i-M-MLV, i-RI), okwenza kube lula ukwakhiwa kwamakhithi angemuva kanye nesistimuukulungiswa, ngaphakathi kokuqinisekisa okulula.
Imininingwane
500T, 50,000T
Izingxenye zekhithi
| Okuqukethwe kwekhithi (Isistimu yokusabela engu-20 μL) | IM-05111-01 | IM-05112-01 |
| 500 T | 50,000 T | |
| Ingxube Ye-enzyme Eqondile | 500 μL×1 | 50 ml×1 |
| 2× Ibhafa ye-RT-qPCR eqondile | 1 mL×5 | 500 ml×1 |
| Isiyalezo | 1 | 1 |
Izinyathelo Zokusebenza
Ngaphambi kokulungiswa kwe-reagent, wonke ama-reagents kukhithi kufanele ancibilikiswe ekamelweni lokushisa futhi axutshwe ngobumnene.
A: Lungiselela of isifanekiso futhi i-reagent
1. Lungiselela ithempulethi ye-RNA elungisiwe (Kutuswa ukusebenzisa ikhithi yochungechunge lwe-Foregene Total RNA Isolation Kit ukuze ukhiphe futhi uhlanze ithempulethi ye-RNA) noma isampula yomkhiqizo wokuqhekeka (Kutuswa ukusebenzisa uhlelo lwe-Foregene Lysis), ama-primers athile(10 μM) nezinye izinto ezisetshenziswayo ezifanele, ithuluzi.
2. Faka i-2× Direct RT-qPCR Buffer, i-ddH2O yamahhala ye-RNase kanye ne-20× ROX Reference Dye (uma kunjalo's okudingekayo) ebhokisini elineqhwa, okwenza lezi zincibilike ngokwemvelo, futhi zixutshwe ngobumnene.
B: Lungiselela of I-RT-qPCR uhlelo
Ithebula 1 : Lungiselela of I-RT-qPCR suhlelo
| Isakhi | Ivolumu | Ukugxila kokugcina |
| 2× Ibhafa ye-RT-qPCR eqondile | 10 μL | 1× |
| Ingxube Ye-enzyme Eqondile | 1μL | |
| I-Primer Phambili(10 μM) | 0.8 μL | 50-900nM |
| I-Primer Ehlehlayo(10 μM) | 0.8 μL | 50-900nM |
| I-Probe(4 μM) | 1μL | 200nM |
| Isifanekiso(i-RNA noma i-Lysate) | X μL | |
| I-20 × ROX Ireferensi Idayi | - | 1* |
| I-RNase-FreeddH2O | (6.4-X) μL | |
| Ivolumu ephelele | 20μL |
C: Setha i I-RT-qPCR ukusabela uhlelo
Ithebula 2 : I-RT-qPCR sabelaneon uhlelo ukulungiselelwa (okwe isibonelo: ezimbili-isinyathelo indlela)
| Isinyathelo | Izinga lokushisa | Isikhathi | Imijikelezo | Okuqukethwe | |
| 1 | 50℃ | 15 imiz | 1* | 1 | Hlehlisa ukuloba |
| 2 | 95℃ | 1 iminithi | 1 | I-pre-denaturation | |
| 3 | 95℃ | 10 isekhondi | 40-45 | I-Denaturation | |
| 60℃ | 30 isekhondi 2* | I-Anneal/Isandiso | |||
Isitoreji kanye Nempilo yeshelufu
-Ikhithi kufanele igcinwe ku -20± 5℃.Isikhathi sokuqinisekisa yizinyanga eziyi-12.
-Gwema imijikelezo yokuqhwaza ephindaphindiwe (<5 imijikelezo).
